Several methods for determining a component in a sample by utilizing oxidoreductase which catalyzes a reaction involving NAD(P)-NAD(P)H are known.
The methods are advantageous in stoichiometrical accuracy, small influence of other components, and the like. However, when NAD(P)H derived from components other than the objective component (hereinafter referred to as other components) is added during the determination, such NAD(P)H must be avoided or removed.
To avoid or remove such NAD(P)H, there is a method in which a reagent blank test is separately carried out and after determination of other components (so called blank test of a sample), the sum of NAD(P)H derived from the other components and the objective components is determined and the amount of the objective component is determined by calculation.
For example, in the case of determination of amylase activity in blood, the enzyme reaction described below is carried out and the rate of formation of NAD(P)H is determined, whereby the amylase activity can be determined. However, as maltose and glucose are contained in blood, NAD(P)H must be determined after they are removed or decomposed. ##STR1##
In the quantitative determination of NAD(P)H by colorimetry of the reaction solution, the absorbancy of the objective component is added to those of the other components, and the determinable range becomes narrow, depending upon the amount of glucose or maltose in a sample. The reason is that the absorbancy which is detectable with a spectrophotometer according to the currently employed technique is limited to a certain range; that is, it is impossible to detect an absorbancy of 3.0 ABS or more with an ordinary spectrophotometer. In view of this aspect, a region in which a measurement value is low is preferred. Further, in the case of reagent blank test with a high absorbancy, reproducibility is poor. Assuming that blank solutions have absorbancies of, for example, A and B (A&lt;&lt;B) respectively, the absorbancy E(E&lt;&lt;B) appearing as the result of reaction is added to form A+E and B+E (A+E&lt;&lt;B+E). When this run is repeated with measurements, the respective variations referred to as As and Bs are obtained (variations in the blank are similarly referred to as Ab and Bb, respectively). From the nature of a spectrophotometer as an apparatus, the percent transmission (T %) is logarithmically converted to absorbancy. Thus, a difference based on the logarithmic function appears on the variation in absorbancy in the case of low absorbancy (high percent transmission) and on the variation in absorbancy in the case of high absorbancy (low percent transmission), and even though the variations in percent transmission are identical, As&lt;Bs (Ab&lt;Bb) (absorbancy=log I.sub.0 /I, wherein I is transmittance). From the foregoing, the difference E is calculated for each of the blanks A and B.
With respect to A: EQU (A+E).+-.As-A.+-.Ab=E.+-.As.+-.Ab
With respect to B: EQU (B+E).+-.Bs-B.+-.Bb=E.+-.Bs.+-.Bb
From As&lt;Bs and Ab&lt;Bb is led, As+Ab&lt;Bs+Bb; that is, when the blank has a large value, the variation becomes large, so that the coefficient of variation obtained by division by E becomes large.
An object of the present invention is to provide a method for quantitative determination of the objective component utilizing an NAD(P)H-forming reaction system without performing a blank test of the sample, by eliminating NAD(P)H formed from the other components or present in the sample by reactions.